PCT/TR01/00027, filed 2 Jul. 2001, designating the United States and published as WO 02/078703; PCT/TR02/00017, filed 19 Apr. 2002, designating the United States and published as WO 02/078704; and U.S. application Ser. No. 10/682,584, filed 9 Oct. 2003, published as US 20040072914 A1, are each incorporated herein by reference in their entirety.
The gene patched-encodes a transmembrane protein acting as a receptor for the hedgehog proteins identified first by their effect on the patterning of tissues during development. When not liganded by hedgehog, the patched protein acts to inhibit intracellular signal transduction by another transmembrane protein, smoothened. Binding of hedgehog to the patched causes relieving of this inhibition. Intracellular signal transduction by the relieved smoothened then initiates a series of cellular events resulting ultimately in alterations of the expressions of the hedgehog target genes and of cellular behaviour. General features of this hedgehog/smoothened pathway of signal transduction, first identified in Drosophila, are conserved in diverse living organisms from Drosophila to Human. However, the pathway gets more complex in more advanced organisms (e.g. presence in human of more than one genes that display significant similarity to the single patched gene of Drosophila). Inactivating mutations of the patched have been found to cause constitutive (ligand-free) signaling through the hedgehog/smoothened pathway. The hedgehog/smoothened pathway overactivity, resulting from mutations of the patched and/or further downstream pathway elements, is found in all basal cell carcinomas (BCCs). The nevoid basal cell carcinoma syndrome (NBCCS) results from patched haplo insufficiency. Patients with the NBCCS, because of an already mutant patched in all cells, develop multiple BCC's as they grow older.
Hedgehog/smoothened signaling is known to be employed for normal functions in several normal tissues and for the maintenance of normal epithelial stem cells (Zhang Y et al (2001) Nature 410:599-604).
Cyclopamine, a steroid alkaloid, has the chemical formula shown below.
It is found naturally in the lily Veratrum californicum and can be obtained by purification from this and other sources. Teratogenicity of these plants on grazing pregnant animals led to the identification of cyclopamine as an active compound (Keeler R. F. (1969) Phytochemistry 8:223-225). How cyclopamine displayed teratogenicity was revealed by the finding that it is an inhibitor of the hedgehog/smoothened signal transduction pathway (Incardona J. P. et al. (1998) Development 125:3553-3562; Cooper M. K. et al. (1998) Science 280:1603-1607).
The sonic hedgehog protein, a member of the hedgehog family of proteins, has been found to induce differentiation of its target cells, including the precursors of ventral cells in the developing central nervous system (Goodrich L. V. et al. (1998) Neuron 21:1243-1257). Inhibition of the hedgehog/smoothened pathway by cyclopamine in the developing chicken brain prevented formation of the ventral cells and caused holoprosencephaly (Incardona J. P. et al. (1998) Development 125:3553-3562; Cooper M. K. et al. (1998) Science 280:1603-1607), the common malformation observed in the lambs of the sheep grazing Veratrum (Binns W. et al. (1963) Am. J. Vet. Res. 24:11641175). Cyclopamine has been reported to inhibit cellular differentiation in other systems as well, including the differentiation of bone marrow cells to erythroid cells (Detmer K. et al. (2000) Dev. Biol. 222:242) and the differentiation of the urogenital sinus to prostate (Berman D. M. et al. (2000) J. Urol. 163:204).